Supravital Staining 5c5b5n

Supravital staining

Sagar Sharma B.SC.MLT 1

Introduction Staining • is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. • Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes. 2

Vital stain • A vital stain in a casual usage may mean a stain that can be applied on living cells without killing them • Vital stains have been useful for diagnostic and surgical techniques in a variety of medical specialties. • In supravital staining living cells that have been removed from an organism, whereas intravital staining is done by injecting or otherwise introducing the stain into the body. 3

Supravital staining • Supravital staining is a method of staining used in microscopy to examine living cells that have been removed from an organism. • supravital stain may have a greater toxicity, as only a few cells need to survive it a short while. • As the cells are alive and unfixed, outside the body, supravital stains are temporary in nature. • Either the smear is prepared from sediment or directly sample is used .

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Procedure • As per the sample taken . • Smear is prepared from sediment or whole sample . • In some cases 1-2 drops of mixture of dyes is taken , sediment is mixed and covered with cover slip and observed under microscope . • Reticulocytes count : 3-4 drops of methyline blue solution+ venous or capillary blood . Mix well and make a thin smear , dry and observe under 5

Types of supravital stain New methyline blue • organic compound of thiazine class of heterocycles. • It is used as a stain and as an antimicrobial agent. • NMB is a staining agent used in diagnostic cytopathology and histopathology. • Typically used for staining immature red blood cells. It is a supravital stain 6

Neutral red and James Green Stain • use for the differentiation of the macrophages and leukocytes from neoplastic and mesothelial cells . • Result : macrophages and leukocytes are brilliant red neoplastic and mesothelial cells are not stained . 7

Brilliant cresyl blue • Brilliant cresyl blue is an example of supravital stain used for counting reticulocyte. • Using Brilliant Cresyl Blue, the reticulocytes will show a "reticular" (meshlike) network. • The mesh like network is of residual rRNA that has not yet been extruded from the cell. 8

Crystal violet • is a triarylmethane dye. • The dye is used as a histological stain and in Gram's method of classifying bacteria. • In laboratory, solutions containing crystal violet and formalin are often used to simultaneously fix and stain cells grown in tissue culture. • It helps to preserve cells and make them easily visible, since most cells are colourless. 9

Methyl violet • Methyl violet is a family of organic compounds that are mainly used as dyes. • Depending on the amount of attached methyl groups, the color of the dye can be altered. • Medical use : as supravital stain .

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Nile blue • Nile blue (or Nile blue A) is a stain used in biology and histology. • t may be used with live or fixed cells, and imparts a blue colour to cell nuclei. • It may also be used in conjunction with fluorescence microscopy to stain for the presence of polyhydroxybutyrate granules in prokaryotic or eukaryotic cells. • With the Nile blue derivative N-EthylNile Blue (EtNBA), normal and premalignant tissues in animal experiments can be distinguished

Nile blue in water. Left to right: pH 0, pH 4, pH 7, pH 10, pH 14.

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Toluidine blue • Toluidine blue is a basic thiazine metachromatic dye with high affinity for acidic tissue components. • It stains nucleic acids blue and polysaccharides purple and also increases the sharpness of histology slide images. • Used in renal pathology and neuropathology. 12

Major Uses of supravital staining in laboratory 1. Reticulocytes count •.

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The most common supravital stain is performed on smear of human blood for reticulocytes by using new methylene blue or brilliant cresyl blue stains. makes it possible to see the reticulofilamentous pattern of ribosomes characteristically precipitated in these live immature red blood cells by the supravital stains.

2.Rate of red cell production from bonemarrow •. By counting the number of reticulocytes

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3.Detection of hemolytic anemia • When there is an increased production of red blood cells to overcome chronic or severe loss of mature red blood cells people often have a markedly high number and percentage of reticulocytes. • A very high number of reticulocytes in the blood can be described as reticulocytosis.

Supravital stain of a smear of human blood from a patient with hemolytic anemia. The reticulocytes are the cells with the dark blue dots and curved linear structures (reticulum) in the cytoplasm. 14

4. Supravital staining of spermatozoa • Supravital staining of human spermatozoa is a useful technique to assess semen quality. • relationship of eosin concentration to the percentage of cells staining live.

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5.early diagnosis of oral mucosal malignancies • Supravital staining of suspicious mucosal lesions in oral cavity helps in the early diagnosis of malignant lesions. • Toludine blue is the dye which is preferred for the purpose of supravital staining.  • This dye is acidophilic in nature and preferentially stains acidic cellular components like DNA & RNA.  • Malignant lesions of mucosa have large amounts of acidic DNA & RNA due to the active cellular proliferation which is occurring there.  16

6.Flouresscence activated cell sorting (FACS) analysis • Supravital staining can be combined with cell surface antibody staining (immunofluorescence) for applications such as FACS analysis.

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References • John P.greer, John Foerster, George M Rodgers, Daniel A. Arber Wintrobe’s Clinical Hematology.11th (ed.), lippincot ,Williams and Wilkins , 2009 • Wikipedia , staining: supravital staining 2013. http://http://en.wikipedia.org/wiki/supravital_staining • Winifred Gray, Gabrijela Kocjan Diagnostic Cytopathology.3rd (ed.), church hill Livingstone , 2010 • drtbalu's otolaryngology, Role of Supravital staining in early diagnosis of oral mucosal malignancies 2012

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